Method for fostering bone formation and preservation

ABSTRACT

A method of inducing bone formation in a subject in need of such inducement comprises the steps of mechanically inducing an increase in osteoblast activity in the subject and elevating blood concentration of at least one bone anabolic agent in the subject. The method steps may be performed in any order, but in sufficient time proximity that the elevated concentration of the anabolic agent and the mechanically induced increase in osteoblast activity overlaps. The method may additionally comprise providing the subject with an elevated blood concentration of at least one antiresorptive agent, wherein the elevated concentration is sufficient to prevent resorption of new bone growth produced due to the osteoblast activity. Use of the method permits targeting of specific bones of the subject for bone production and preservation, faster bone production and earlier discontinuation of bone anabolic pharmaceuticals. Kits adapted for performing the method are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention is based on and claims priority to ProvisionalApplication No. 60/571,200 which was filed May 14, 2004, the contents ofwhich are specifically incorporated herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The invention disclosed herein was made with Government support underNIH Grant No. DE 12110 from the National Institutes of Health.Accordingly, the government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to a method for fostering boneformation in a subject. More particularly the invention concerns amethod for inducing rapid bone formation and then preserving the bonethus produced, e.g., by minimizing its resorption. The invention permitsspecific targeting of particular bones of a subject for repair,strengthening, reshaping and/or modeling. The invention is additionallydirected to a kit for carrying out the method of the invention.

BACKGROUND OF THE INVENTION

Bones are multi-purpose structures that play diverse, vital roles invertebrates. They provide a framework that supports the body and givesit shape. Bone undergoes a continuous renewal or remodeling during thelifetime of an individual. Bone consists of living cells widelyscattered within a non-living material known as matrix. Two main typesof cells are responsible for bone remodeling: the osteoblasts involvedin bone formation and the osteoclasts involved in bone resorption. Thematrix is formed by the action of osteoblasts, that make and secretebone matrix proteins such as collagen, which provide elasticity, as wellas mineral salts formed from calcium and phosphorous, which imparthardness to bone. As bone tissue matures, some osteoblasts are trappedin the bone matrix and differentiate into osteocytes, which are maturebone cells that carry out normal cellular activities. These osteocytesconnect with other osteocytes through the bone matrix and can sensepressure or cracks in the bone. They therefore assist in directing whereosteoclasts will act to dissolve bone during the repair and/orregeneration of bone.

Osteoclasts are cells that dissolve existing bone, thus facilitatingbone growth, repair and regeneration. Osteoclasts are multinucleatedcells that originate from the fusion of mononuclear phagocytes.Osteoclasts secrete protons that lower the pH of an extracellularcompartment located between osteoclasts and bone. This low pHfacilitates the dissolution of bone crystals and activates lysosomalenzymes that digest the bone matrix. Osteoclasts are therefore powerfuland efficient bone resorbing cells that cover only 0.5% of the bonesurface. With regard to bone formation, osteoblasts produce a structure,known as “osteoid”, which is formed of bone collagen and other proteins.The osteoblasts thereafter control the deposition of calcium and otherminerals into the osteoid in order to produce the calcified bone tissue.Upon the completion of bone formation, the osteoblasts flatten out andform a lining upon the surface of the bone. These flattened osteoblasts,known as “lining cells”, regulate passage of calcium into and out of thebone. In addition, they produce, upon hormonal activation, proteins thatpromote osteoblast differentiation and activation. Making new bone istherefore a slow process that requires the lying down of the osteoid,its maturation and then its calcification. In contrast to osteoclasts,osteoblasts cover 30% of the bone surface.

The bones of the skeleton are not entirely solid throughout. Theoutside, i.e., cortical, bone is substantially solid throughout, havingonly a few small canals. Located inwardly from the cortical bone,however, is spongy bone known as cancellous bone. The cancellous bone iscomposed of a honeycomb network of trabecular bone defining a pluralityof spaces or cavities filled with fluid bone marrow, stem cells and somefat cells. Existing within these bone marrow cavities are, inter alia,various highly specialized cells which assist in breaking down existingbone and correspondingly producing new bone to replace that which isbroken down or which may be otherwise lost due to injury or illnessessuch as osteoporosis.

The physical structure of bone may be compromised by a variety offactors, including disease and injury. One of the most common bonediseases is osteoporosis, which is characterized by low bone mass andstructural deterioration of bone tissue, leading to bone fragility andan increased susceptibility to fractures, particularly of the hip, spineand wrist. Osteoporosis develops when bone resorption occurs toorapidly, if bone replacement occurs too slowly, or due to a combinationof both. This is in part due to the fact that it requires six months forosteoblasts to rebuild the amount of bone destroyed by osteoclasts inthree days. Bone injury, on the other hand, involves localized trauma tothe bone.

A variety of methods are well-known in the art for fostering boneformation in individuals who (1) suffer from diminished bone mass due,for example, to illness, (2) are subjected to bone trauma causing injurysuch as bone fractures, and (3) need to strengthen bone, such asvertebral bones. Such prior art methods for treating these disorders aretypically systemic in nature, however. That is, they treat the wholeskeleton as a single entity. These methods are therefore not commonlyable to be targeted on one or more specific bones, e.g., those of thehip, shoulder, spine and/or wrist, which may require a more focusedtreatment due to bone losses due to disease effects caused by, e.g.,osteoporosis or by bone trauma such as that due to a fracture. Moreover,prior art methods frequently require undesirably long treatmentregimens, with accompanying patient compliance problems.

There has thus been a long-felt need by those working in this field fora faster and more targeted method of inducing bone formation in subjectssuffering from diminished bone mass, especially for a method coupledwith an enhancement in retention of the new bone so produced. Thepresent invention permits, in addition to the general systemic effectnoted above, specific targeting of one or more particular bones or bonyareas most in need of such treatment for rapid bone formation. Asexplained below, the method and kit of the present invention areparticularly adapted to provide more effective bone formation withincreased rapidity while permitting the retention of the bone thusproduced and thus to admirably fulfill the desired functions.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a methodfor inducing rapid bone formation in a subject such that the length oftreatment with a bone anabolic agent may be reduced, thus offeringsafety and pharmacoeconomic advantages.

It is also an object of the present invention to provide a method forinducing bone formation at a rapid rate in a subject suffering fromdiminished bone mass which permits specific bones of the subject to betargeted for an increase in bone mass, while additionally providingbeneficial bone formation effects to the entire skeleton of the subject.

It is another object of the invention to provide a method capable ofinducing rapid bone formation in subjects so as to prevent and/or treatbone fractures.

It is a further object of the invention to provide a method capable ofinducing rapid bone formation in subjects requiring such additional boneto serve as an anchoring mechanism for prostheses of, e.g., the hip,knee and shoulder and/or other types of implants such as dentalimplants.

It is a still further object of the invention to provide a methodcapable of inducing rapid bone formation in subjects requiring bonestrengthening to alleviate chronic pain due to conditions such asvertebral crush.

It is yet another object of the invention to provide a method capable ofdiminishing resorption of any substantial portion of such additionalbone thus produced in accordance with the invention.

It is another object of the invention to provide a method capable ofinducing rapid bone formation so as to permit bone reshaping/modelingvia said additional bone formation.

It is a further object of the invention to provide an article ofmanufacture comprising a kit adapted to enable one to carry out theabove-described method of the invention.

In a preferred embodiment, the method of the invention is utilized witha human subject. However, the invention additionally contemplatesveterinary applications.

In one embodiment, the invention provides a method of inducing boneformation in a subject in need of such inducement comprising the stepsof (a) mechanically inducing an increase in osteoblast activity in thesubject; and (b) elevating blood concentration of at least one boneanabolic agent therein, e.g., by administering such an agent or byadministering a compound which causes natural formation of such anagent. The aforesaid steps may be performed in any order, but insufficient time proximity that the elevated concentration of theanabolic agent and the mechanically induced increase in osteoblastactivity at least partially overlaps.

In a further embodiment of the invention the method comprises the stepsof targeting for treatment at least one bone of the subject, whereineach such targeted bone defines a bone marrow cavity therein. The bonemarrow cavity contains, inter alia, a quantity of bone marrow and aplurality of osteoblasts. The method further comprises mechanicallyaltering the contents of the bone marrow cavity to thereby stimulate andthus increase osteoblast differentiation and/or activity therein. Themethod additionally comprises administering to the subject at least onebone anabolic agent for a duration and at a concentration sufficient toraise blood levels of the anabolic agent within the subject abovenatural levels thereof and thereby prolong the mechanically inducedosteoblast activity. The mechanical alteration of the contents of thebone marrow cavity in bones wherein it is desired to foster such bonegrowth permits specific bones of the subject to be particularly targetedfor inducing bone formation therein.

The invention further provides a method of inducing bone formation in asubject suffering from diminished bone mass which comprises the steps oftargeting for treatment at least one bone of the subject, wherein eachtargeted bone defines a bone marrow cavity therein. The bone marrowcavity contains a quantity of bone marrow and a plurality ofosteoblasts. The method of the invention further comprises mechanicallyaltering the contents of the bone marrow cavity to thereby stimulate andthus increase osteoblast activity (e.g., increased differentiation orincreased bone formation by stimulating osteoblasts) therein.Thereafter, bone mass is increased within the cavity due to theincreased osteoblast activity. The method additionally comprisesadministering to the subject at least one bone anabolic agent for aduration and at a concentration sufficient to raise blood levels of theanabolic agent within the subject above natural levels thereof andthereby prolong the mechanically induced osteoblast activity. Themethod, however, further comprises additionally administering, either(1) contemporaneous with, (2) overlapping with, or (3) subsequent to theadministration of the bone anabolic agent, an antiresorptive agent for aduration and at a concentration sufficient to diminish resorption of newbone produced due to the osteoblast activity. As with the methoddescribed above, the mechanical alteration of the contents of the bonemarrow cavity of bones in which such stimulated growth is desired thuspermits specific bones of the subject to be particularly targeted forenhanced bone formation.

The invention additionally provides a kit for fostering bone formationin at least one targeted bone of a subject in need of such boneformation. The kit includes at least one container having therein atleast one bone anabolic agent, as well as a mechanical alteration devicefor altering contents of a bone marrow cavity in at least one targetedbone. The kit may additionally be provided with an evacuation device forevacuating at least a portion of the contents from the bone marrowcavity.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIG. 1 is a graphical representation of the results of a PIXIMUSanalysis of the femur distal marrow cavity bone mineral density (“BMD”)of groups of laboratory rats tested at one-week intervals during athree-week test regimen. The PIXIMUS analyzer is available from LunarCorp., Palo Alto, Calif. and provides Dual Energy X-Ray Absorptiometry(“DEXA”) data for small animals such as mice and rats. MaleSprague-Dawley rats (5 animals per group) aged 9 weeks were sacrificedand tested at time 0 and after 1, 2 and 3 weeks. The various groups weresubjected to the following treatment modalities: Group A—left femurcontrol; Group B—left femur sham; Group C—mechanical bone marrowablation (bmx) to alter the contents of the bone marrow cavity of theleft femur; Group D—mechanical bone marrow ablation treatment (bmx) ofthe left femur, coupled with the administration of an amidatedparathyroid hormone truncate (PTH[1-34]-NH₂) during days 1-21; and GroupE—a right (non-bmx) femur BMD measurement of rats treated during days1-21 with an amidated parathyroid hormone truncate (PTH[1-34]-NH₂)wherein the rat's left femur had undergone a bone ablation (bmx)treatment;

FIG. 2 is a graphical representation of the results of a PIXIMUSanalysis of the femur distal marrow cavity BMD of a second set of groupsof male Sprague-Dawley rats (5 animals per group) aged 9 weeks whichwere sacrificed and tested at time 0 and after 1, 2 and 3 weeks. Thevarious groups were subjected to the following treatment modalities:Group F—left femur control; Group G—left femur sham; Group H—mechanicalbone marrow ablation (bmx) to alter the contents of the bone marrowcavity of the left femur; Group I—mechanical bone marrow ablation (bmx)of the left femur, coupled with administration of an amidatedparathyroid hormone truncate (PTH[1-34]-NH₂) during days 1-7, followedby the administration of a salmon calcitonin (sCT) antiresorptive agentduring days 7-21; and Group J—a right femur BMD measurement of ratstreated with an amidated parathyroid hormone truncate (PTH[1-34]-NH₂)wherein the rat's left femur had undergone a bone marrow ablation (bmx)treatment;

FIGS. 3A-3D illustrate a representative series of steps for carrying outa left femur bone marrow ablation in a rat from one of Groups A-Jdiscussed above. FIG. 3A) accessing the distal femur; FIG. 3B) drillinginto the bone marrow cavity of the distal femur; FIG. 3C) washing thebone marrow cavity; and FIG. 3D) suturing the incision;

FIG. 4 is a bar graph comparing the Lumbar Vertebral Bone MineralDensity obtained in osteoporotic female Sprague-Dawley rats wherein theosteoporosis was induced via an ovariectomy. The rats were treated with(1) PTH[1-31]-NH₂; (2) PTH[1-34]-NH₂; and PTH[1-34]-OH; and

FIG. 5 is a bar graph comparing the Lumbar Vertebral Bone Formation Rateobtained in osteoporotic female Sprague-Dawley rats wherein theosteoporosis was induced via an ovariectomy. The rats were treated with(1) PTH[1-31]-NH₂; (2) PTH[1-34]-NH₂; and PTH[1-34]-OH.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

In a first embodiment the invention provides a method of inducing newbone formation in a subject in need of such inducement. The methodcomprises the steps of mechanically inducing an increase in osteoblastactivity in the subject and elevating blood concentration of at leastone bone anabolic agent in the subject. The above steps may be performedin any order, but are to be carried out in sufficient time proximitythat the elevated concentration of the anabolic agent and themechanically induced increase in osteoblast activity at least partiallyoverlap.

Inducement of bone growth may include, for example, generating new oradditional bone at locations where such bone growth is not presentlytaking place and/or stimulating the growth (i.e., increasing therapidity thereof) of bone which is already in the process of formation.Without being bound in any way by theory, applicants believe that theinducement of bone growth takes place due to the combined effects of (1)a mechanical inducement of osteoblast activity in the subject coupledwith (2) an elevation in the blood concentration of at least one boneanabolic agent therein.

Mechanical inducement of an increase in osteoblast activity may beobtained, in a preferred embodiment of the invention, by a process ofbone marrow irrigation and ablation. Again, without being bound in anyway by theory, applicants believe that the bone marrow irrigation andablation process leads to the formation of a clot within the bone marrowcavity which, through a cascade of biochemical reactions, contributes toincreasing osteoblast activity in the subject.

In an another embodiment, the increased osteoblast activity mayalternately be obtained by coupling the mechanical inducement with anadditional form of inducement such as biochemical inducement. Suchbiochemical inducement may be obtained by administering to the subject,for example, a quantity of a blood factor such as Factor (“F”) VII,Factor VIIa or a combination thereof. Following tissue or vascularinjury clotting is initiated by the binding of plasma FVII/FVIIa totissue factor (tissue thromboplastin). This complex(FVII/FVIIa+Thromboplastin) initiates a sequence of events which leadsto activation of the coagulation cascade ultimately leading to fibrindeposition and platelet activation. This complex sequence of events maycontribute in part to the stimulation of osteoblasts in the bone marrow.Factors VII and VIIa are commercially available from Novo Nordisk.

The increase in osteoblast activity obtained with the use of the methodof the invention may be due to a variety of factors including, but notnecessarily limited to (1) osteoblast differentiation, i.e., theproduction of additional osteoblasts, (2) increasing the activity and/oreffectiveness of osteoblasts which are already present in inducing boneformation in the subject, and (3) a combination thereof. In a preferredembodiment of the invention, the increase in osteoblast activity wouldinclude all of the above-noted functions.

In one embodiment, the blood concentration of the at least one boneanabolic agent may be elevated by direct administration of one or morebone anabolic agents to the subject.

In a further embodiment of the invention the method additionallycomprises “targeting” one or more specific bones of the subject forinducement of bone growth. This targeting is accomplished bymechanically altering the contents of a bone marrow cavity within eachtargeted bone so as to induce the increased osteoblast activity therein.

The method of the invention is thus useful not only for bone repair,i.e., as in the case of a bone fracture due to trauma, but also forstrengthening bone in a site-specific manner in the case of individualsshown by Dual Energy X-Ray Absorptiometry (“DEXA”) or other techniquesto require an increase in bone mass and/or density to prevent bonefractures, or who suffer due to bone weakness from chronic painattributable to conditions such as vertebral crush. Moreover, as notedabove, the method of the invention additionally serves to provide (andretain) new bone needed to serve as an anchor for prostheses such asartificial hips, knees and shoulders and/or for implants such as dentalimplants.

In a still further embodiment, the method of the invention additionallycomprises providing the subject with an elevated blood concentration ofat least one antiresorptive agent, wherein the elevated concentration issufficient to diminish resorption of new bone growth produced due to themechanically induced enhanced osteoblast activity according to theinvention.

In one embodiment the invention provides a method of inducing boneformation in a subject in need of the same, wherein the method comprises(a) mechanically inducing an increase in osteoblast activity in thesubject; and (b) administering to the subject at least one agent thatcauses elevated blood levels of an endogenous bone anabolic agent withinthe subject. The method steps may be performed in any order, but insufficient time proximity that the elevated concentration of theanabolic agent and the mechanically induced increase in osteoblastactivity at least partially overlaps. In one embodiment of the method,the agent causing an increased expression of the endogenous boneanabolic agent may be a calcilytic agent. Calcilytic agents useful withthe method of the invention include, but are not limited to any agentthat limits the binding of calcium to its receptor and thereby triggersthe release of endogenous PTH. Examples of such calcilytic compounds areset forth in U.S. Pat. Nos. 6,362,231; 6,395,919; 6,432,656 and6,521,667, the contents of which are incorporated herein by reference.

The invention additionally provides a method of inducing bone formationin a subject suffering from diminished bone mass or bone trauma. Themethod includes the step of targeting for treatment at least one bone ofthe subject, wherein each targeted bone defines a bone marrow cavitytherein. The bone marrow cavity contains, inter alia, a quantity of bonemarrow and a plurality of osteoblasts. The method of the inventionfurther comprises mechanically altering the contents of the bone marrowcavity to thereby stimulate and thus increase osteoblast activitytherein. The method additionally comprises administering to the subjectat least one bone anabolic agent for a duration and at a concentrationsufficient to raise blood levels of the anabolic agent within thesubject above natural levels thereof and thereby prolong themechanically induced osteoblast activity. For example, a bone anabolicagent endogenously produced in the human body is PTH[1-84] in the freeacid form which is naturally found in levels of less than about 8picomoles (pmoles) per liter in blood of a human subject. Thus thepractice of the invention would involve, as indicated above, raising theblood levels of the bone anabolic agent within the subject to levelscorrespondingly above such natural level. Further to the above, themechanical alteration of the contents of the bone marrow cavity thuspermits specific bones of the subject to be targeted for enhanced boneformation.

In an embodiment of the invention, bone formation may be induced at alocation of a long bone fracture in bone of the subject to increase therapidity of healing of the fracture. In another embodiment, the methodfurther comprises reshaping or modeling at least one targeted bone of asubject by inducing additional bone formation in a controlled mannerthereon.

Applicants have surprisingly discovered that an initial burst ofosteoblast activity can be sustained at unexpectedly high levels byelevating blood levels of a bone anabolic agent during a time that atleast partially overlaps with the initial burst of osteoblast activity.This effect is demonstrated by comparing the curves obtained with GroupsC and D in FIG. 1. Absent the bone anabolic agent, the initial burst ofbone formation is quickly followed by the resorption of newly formedbone (see, e.g., the Group C curve in FIG. 1). The curve obtained withGroup E of FIG. 1 shows that although increased bone growth is induceddue to the administration of a PTH bone anabolic agent alone, thisincrease does not rise to the level achieved due to the coupling of bonemarrow ablation (bmx) plus administration of PTH. The results set forthin FIG. 1 clearly demonstrate that the coupling of bmx and PTHadministration provides an unexpected increase in degree, speed andlongevity of bone formation relative to the effects of bmx or PTH alone,as well as a targeted site-specific response. Table 1 (below) sets forththe values upon which the curves obtained with Groups A-E in FIG. 1 arebased. As no readings were taken for the Control (Group A) and Sham(Group B) groups at Week 1 and Week 2, these values were estimated forpurposes of preparing the curves for these two Groups. Table 1,moreover, provides the standard deviations for the values obtained ineach Group, which standard deviations are also indicated in FIG. 1.

TABLE 1 BMD (DEXA) (gm/cm²) ± standard deviation Groups Femur Week 0Week 1 Week 2 Week 3 Control (Group A) LEFT 0.096 ± .007 0.105 0.1150.124 ± .009 Sham (Group B) LEFT 0.096 ± .007 0.1 0.107 0.113 ± .006 BMXALONE (Group C) LEFT 0.099 ± .009 0.140 ± .020 0.115 ± .006 0.124 ± .006BMX + PTH 1-21 LEFT 0.096 ± .007 0.166 ± .004 0.200 ± .030 0.190 ± .008(Group D) PTH 1-21 (Group E) RIGHT 0.097 ± .008 0.124 ± .007 0.141 ±.007 0.154 ± .007

Peripheral Quantitative Computerized Tomography (“pQCT”) measurementstaken by the inventors during the above-discussed experiments did notdemonstrate an improvement in total bone content for the combination ofbmx+PTH relative to PTH alone. This is due to the fact that the femurcontains mostly cortical bone and the pQCT data (which has not yet beenbroken out into values corresponding to the various types of bone)presently is directed to an aggregate of all of the bone tissues.

FIG. 2 is provided to illustrate, inter alia, the further beneficialeffect achieved due to the overlapping (i.e., with PTH administration)or subsequent administration of the antiresorptive agent salmoncalcitonin (sCT). First, a comparison of the curves obtained with GroupH (bmx alone) and Group J (PTH+sCT with no bmx) to that obtained withGroup I (bmx+PTH+sCT) provide still further evidence of the synergisticeffect attributable to the use of the method of the invention.Additionally, the curve obtained with Group I also demonstrates that theadministration of the antiresorptive agent sCT following the initialburst of osteoblast activity which occurs due the coupling of bmx andPTH administration, significantly diminishes the proportion of the newlyformed bone which is adsorbed into the subject's body due to the actionof the osteoclasts. Table 2 (below) sets forth the values upon which thecurves of FIG. 2 are based. As no readings were taken for the Control(Group F) and Sham (Group G) during Weeks 1 and 2, these values wereestimated for purposes of preparing the curves. Table 2 provides thestandard deviations for the values obtained in each Group, whichstandard deviations are also indicated in FIG. 2.

TABLE 2 BMD (DEXA) (gm/cm²) ± standard deviation Groups Femur Week 0Week 1 Week 2 Week 3 Control (Group F)* LEFT 0.096 ± .007 0.105 0.1150.124 ± .009 Sham (Group G) * LEFT 0.096 ± .007 0.1 0.107 0.113 ± .006BMX ALONE (Group H)* LEFT 0.099 ± .009 0.140 ± .020 0.115 ± .006 0.124 ±.006 BMX + PTH 1-7 + LEFT 0.096 ± .007 0.166 ± .004 0.165 ± .017 0.157 ±.014 sCT 7-21 (Group I) PTH 1-7 + sCT 7-21 RIGHT 0.097 ± .008 0.124 ±.007 0.128 ± .003 0.140 ± .007 (Group J) *Groups F, G and H are the sameas Groups A, B and C (see Table 1).

The results set forth in FIGS. 1 and 2 and in corresponding Table 1 andTable 2 discussed above were obtained with the use of a method formechanically inducing increased osteoblast activity as illustrated inFIGS. 3A-3D. The method shown in FIGS. 3A-3D is provided only for thepurpose of illustration, however, and the invention is not limited tothe procedure(s) illustrated in the indicated Figures since a number ofalternate techniques, which would be well known to those of ordinaryskill in the art, may be used to obtain the necessary mechanicalinduction of osteoblast activity. Thus, in the representative methodillustrated in FIGS. 3A-3D, Male Sprague Dawley rats, 9 weeks old, i.e.,at a rapid growth stage, were housed for 5-10 days prior to surgery(bmx). On the day of surgery, the rats were weighed and anesthetizedwith a combination of ketamine (50 mg/kg) and xylazine (10 mg/kg) usinga decapione. The anesthetic was prepared by combining 0.4-0.8 mlKetamine with 0.5 ml. Xylazine and 8.5 ml saline. The rats were eachinjected with the anesthetic at the rate of 0.1 ml/10 g body weight.

In preparation for surgery, the hair over the left knee joint of eachrat was shaved. The shaved area was prepped with Betadine scrub and thenwashed with ethanol. A longitudinal 1.0 cm. incision was then madeacross the medial aspect of the knee joint (see FIG. 3A). The knee wasthen bent and the incised tissue was pulled back to expose the patellatendon as a landmark. The patella was pushed aside and a 1.0 mm hole wasdrilled through the femoral intracondylar notch above the tendon by asmooth 0.035 k-wire drill bit into the marrow cavity (see FIG. 3B). Thedrilling motion was repeated 5 times. Subsequently, the above-describeddrilling step was repeated using a threaded 0.045 inches drill bit.

Once the hole was completed, the contents of the bone marrow cavity wereback-flushed by injecting 5 ml of normal saline solution into the femurusing a syringe attached to a 26-gauge needle (see FIG. 3C). The nextstep involved suturing the medial ligamentous structures with 4-0 Dexonthread and closing the skin incision with surgical metal clips (see FIG.3D). Thereafter, each rat was injected with a 5 ml bolus of saline andtagged for identification. During recovery, the rats were given TYLENOL®brand acetaminophen solution (300 mg/kg/day) for the first 24 hoursafter surgery and then checked on a daily basis for the first 5 days.

On the day of sacrifice, each rat was euthanized in a CO₂ chamber. Therat's blood was then collected via a cardiac puncture. The femurs wereremoved and fixed in a 4% formalin solution, after which they weredehydrated in a graded series of ethanol solutions.

The bones were thereafter subjected to a variety of sample analysistechniques. These included X-ray analysis wherein the femurs wereX-rayed in individually sealed plastic bags; PIXIMUS analysis using aLunar PIXIMUS scanner to measure bone mineral density in the bone marrowcavity of the femurs; MicroCT analysis; and blood analysis forosteocalcin, PTH, CT, NTX and growth factors.

In one embodiment of the invention, the bone anabolic agent may beadministered to the subject contemporaneous with the mechanicalinducement of osteoblast activity (whether by increased osteoblastformation and/or by increased bone formation by pre-existingosteoblasts), which mechanical inducement may be achieved, e.g., byalteration of the bone marrow cavity. In preferred embodiments, marrowand/or other components of the marrow cavity is/are removed underpressure (e.g., by altering the relative pressure within versus withoutthe marrow cavity).

In another embodiment the bone anabolic agent is administered subsequentto such mechanical inducement. In another embodiment the bone anabolicagent may be administered prior to mechanical inducement such thatelevated levels of bone anabolic agent are already present at the timeof mechanical inducement, which levels may then be maintained orcontinued intermittently for an extended period thereafter.

The bone anabolic agent may be administered orally, intravenously,intramuscularly, subcutaneously, via implant, transmucosally,transdermally, rectally, nasally, by depot injection or by inhalationand pulmonary absorption. In another embodiment the bone anabolic agentmay be administered once as a time release formulation, a plurality oftimes, or over one or more extended periods. It is preferred thatelevated blood levels of the anabolic agent be maintained at leastintermittently for between about 14-365 days, and more preferably forbetween about 30-180 days, post-mechanical induction. Intermittentadministration of parathyroid hormone, e.g., PTH[1-34]-NH₂, could occuronce daily or once weekly resulting in peaks of blood concentration thatreturn to baseline levels between doses, but nevertheless result inperiodic elevated blood levels of a bone anabolic agent in a manner thatoverlaps the elevated osteoblast activity that is initially inducedmechanically, although thereafter sustained, at least in part, by theanabolic agent.

In an additional embodiment the anabolic agent is selected from thegroup consisting of a parathyroid hormone (PTH), anabolic Vitamin Danalogs, a low-density lipoprotein receptor-related protein 5 (LRP5), anactivator of non-genomic estrogen-like signaling (ANGELS), a bonemorphogenic protein (BMP), an insulin-like growth factor (IGF), afibroblast growth factor (FGF), sclerostin, leptin, a prostaglandin, astatin, strontium, a growth hormone, a growth hormone releasing factor(GHRF), hepatocyte growth factor (HGF), calcitonin gene related peptide(CGRP), parathyroid hormone related peptide (PTHrP), transforming growthfactor (TGF)-β1 and combinations thereof. As used herein, the termparathyroid hormone includes, but is not limited to natural parathyroidhormone, a truncate of natural parathyroid hormone, an amidated truncateof natural parathyroid hormone, an amidated natural parathyroid hormoneand combinations thereof.

In one embodiment the bone anabolic agent is truncated PTH[1-34]in thefree acid form. This material is commercially available in anFDA-approved pharmaceutical formulation from Eli Lilly & Co. under thetrade mark FORTEO® (teriparatide). Other useful bone anabolic agents foruse with the invention include, but are not limited to, an amidatedtruncate of natural parathyroid hormone, PTH[1-30]NH₂, PTH[1-31]NH₂,PTH[1-32]NH₂, PTH[1-33]NH₂,PTH[1-34]NH₂ and combinations thereof. In onepreferred embodiment the bone anabolic agent is PTH[1-34]NH₂. Methodsfor the preparation of truncated parathyroid hormones are described inU.S. Pat. No. 6,103,495 to Mehta et al. Moreover, methodologies foramidating such truncated parathyroid hormones are provided in, forexample, U.S. Pat. No. 5,789,234 to Bertelsen et al. and U.S. Pat. No.6,319,685 to Gilligan et al. The contents of each of these patents isspecifically incorporated herein by reference.

In one embodiment of the present method, a sufficient amount of thepreferred truncated parathyroid hormone as discussed herein isadministered to the subject to achieve, and thereafter maintain, apulsatile blood concentration thereof in the subject of between about 50and 350 pg/ml, preferably between about 100 and 200 pg/ml, and mostpreferably about 150 pg/ml. In another embodiment, the bloodconcentration of the parathyroid hormone in the subject is raised to itspreferred level by no later than 7 days following mechanical alterationof the contents of the bone marrow cavity. As would be well known inthis art, an appropriate dosage of the PTH bone anabolic agent must becalculated to achieve the above-indicated blood concentrations. In thecase of injectable formulations, for example, the dose (in pure weightof the active hormone) given to, for example, a human subject, may bethat taught in the literature relating to the bone anabolic activity ofthese various agents. Such dose may, but does not necessarily, rangebetween about 10-200 μg, given once per day, more preferably betweenabout 20-100 μg per dose and most preferably between about 20-50 μg perdose. Dosage levels of injectable formulations comprising bone anabolicagents other than the above-described parathyroid hormone-based agentswould be consistent with those noted above for the PTH agents.

A series of experiments was performed comparing (1) lumbar vertebralbone mineral density, and (2) lumbar vertebral bone formation rateachieved with PTH[1-31]-NH₂ and PTH[1-34]-OH compared to the preferredPTH[1-34]-NH₂ analog discussed above in the absence of the mechanicalinduction step of the invention. The study involved a four-weektreatment of female Sprague-Dawley rats at 10 months of age, and 6months post-ovariectomy (“OVX”), which induces osteoporosis, with one ofthe three above-noted parathyroid hormone truncates. The rats wererandomized into the following groups: sham OVX, OVX+vehicle, OVX+PTH[1-31]-NH₂ or PTH [1-34]-NH₂ (obtained from Unigene Laboratories, Inc.)at 2.5, 10 or 40 μg/kg/day subcutaneous, or PTH[1-34]-OH (obtained fromBachem) at 10 μg/kg/day subcutaneous. After 4 weeks of treatment, theright femur of each animal was analyzed by DEXA and bonehistomorphometry. FIG. 4 is a bar graph providing a comparison of thelumbar vertebral bone mineral density achieved with PTH[1-31]-NH₂,PTH[1-34]-NH₂ and PTH[1-34]-OH. FIG. 5 is a bar graph comparing thelumbar vertebral formation rates achieved with these same truncates.Based on the results of these experiments it was determined that thebone anabolic activity of each of these analogs of PTH is substantiallyequivalent. Because of the similarity of anabolic action among theabove-discussed parathyroid hormones, it is therefore reasonable toexpect that all known PTH analogs will advantageously function in thedesired manner in the method of the present invention.

In a further embodiment of the invention the mechanical induction ofosteoblast activity is accomplished by inserting, into a bone marrowcavity of a bone targeted for enhanced bone formation, an objectconfigured or adapted to physically alter the contents of the cavity andthereby to stimulate the osteoblast activity within the cavity. Inanother embodiment the mechanical alteration may include removal of atleast a portion of the cavity contents.

In a still further embodiment, the method of the invention additionallycomprises administering to the subject an antiresorptive agent for atime and at a concentration sufficient to substantially preventresorption of the new bone produced due to the osteoblast activity. Inone embodiment the antiresorptive agent may be administeredcontemporaneous with the administration of the bone anabolic agent. Inanother embodiment the antiresorptive agent is administered subsequentto the administration of the bone anabolic agent. In a furtherembodiment the administration of the antiresorptive agent may becommenced during administration of the bone anabolic agent and suchadministration may then be continued beyond the termination ofadministration of the bone anabolic agent.

In another embodiment of the invention a single agent may byadministered having both bone anabolic and antiresorptive properties.Examples of such materials include, but are not limited to estrogen,strontium ranalate and selective estrogen receptor modulators (SERMS).

In an embodiment of the method of the invention the antiresorptive agentmay be a calcitonin selected from the group consisting of humancalcitonin, salmon calcitonin (“sCT”), eel calcitonin, elkatonin,porcine calcitonin, chicken calcitonin, calcitonin gene related peptide(CGRP) and combinations thereof. In a preferred embodiment theantiresorptive agent is salmon calcitonin. Blood levels of calcitonin,when used as the antiresorptive agent, preferably range between about5-500 pg/ml, more preferably between about 10-250 pg/ml and mostpreferably 20-50 pg/ml. Moreover, human dosage levels of the subjectcalcitonin agents necessary to achieve the above blood levels, in thecase of, e.g., injectable formulations, may be those taught in theliterature relating to the use of these materials as anabolic agents.Such dose may, but does not necessarily, range between about 5-200 μg/kggiven once per day, more preferably between about 5-50 μg/kg and mostpreferably 8-20 μg/kg by weight of the pure drug, administered daily.Salmon calcitonin (sCT) administered by alternate routes, i.e., by nasalor oral administration, would require higher dosages than thosediscussed above.

Alternately, a variety of additional antiresorptive agents (i.e., otherthan the calcitonins) are useful with the method of the presentinvention. These include, generally, hormone replacement therapy (HRT)agents such as selective estrogen receptor modulators (SERMS),bisphosphonates, cathepsin-K inhibitors, strontium ranalate and variouscombinations thereof. Specific examples of additional antiresorptiveagents include, but are not limited to, (1) PREMARIN® available fromWyeth Laboratories, which includes estrogen as the active ingredient. Atypical accepted dosage is one 0.625 mg tablet daily; (2) ACTONEL®available from Proctor & Gamble, which includes, as its activeingredient, risedronate sodium. A typical accepted dosage is one 5 mgtablet daily or one 35 mg tablet weekly; (3) EVISTA® sold by Eli Lilly &Co. which includes raloxifene HCl as the active ingredient. A typicalaccepted dosage of this formulation is one 60 mg tablet taken daily; and(4) FOSAMAX® available from Merck Pharmaceuticals, which includesalendronate as the active ingredient. Typical dosages of this materialare 10 mg/day or 70 mg/week.

Except where otherwise noted or where apparent from the context, dosagesherein refer to the weight of the active compounds unaffected bypharmaceutical excipients, diluents, carriers or other ingredients,although such other ingredients are typically included in the variety ofdosage forms useful in the method of the invention. Any dosage form(i.e., capsule, tablet, injection or the like) commonly used in thepharmaceutical industry is appropriate for use herein and the terms“excipient”, “diluent” or “carrier” include such non-active ingredientsas are typically included, together with active ingredients, in theindustry. For example, typical capsules, pills, enteric coatings, solidor liquid diluents or excipients, flavorants, preservatives, or the likeare included. Moreover, it is additionally noted that with respect toall of the dosages recommended herein, the attending clinician shouldmonitor individual patient response, and adjust the dosage accordingly.

The antiresorptive agent may be administered orally, intravenously,intramuscularly, subcutaneously, via implant, transmucosally, rectally,nasally, by depot injection, by inhalation and pulmonary absorption ortransdermally. Moreover, the antiresorptive agent may be administeredonce, a plurality of times, or over one or more extended periods. Theantiresorptive agent may be administered orally, intravenously,intramuscularly, subcutaneously, via implant, transmucosally, rectally,nasally, by depot injection, by inhalation and pulmonary absorption ortransdermally. Moreover, the antiresorptive agent may be administeredonce, a plurality of times, or over one or more extended periods.

The invention additionally provides a method of inducing bone formationin a subject suffering from diminished bone mass which comprises thesteps of targeting for treatment at least one bone of the subject,wherein each targeted bone defines a bone marrow cavity therein. Thebone marrow cavity contains a quantity of bone marrow and a plurality ofosteoblasts. The method of the invention further comprises mechanicallyaltering the contents of the bone marrow cavity to thereby stimulate andthus increase osteoblast activity therein. Thereafter, bone mass isincreased within the cavity due to the increased osteoblast activity.The method additionally comprises administering to the subject at leastone bone anabolic agent for a duration and at a concentration sufficientto raise blood levels of the anabolic agent within the subject abovenatural levels thereof and thereby prolong the mechanically inducedosteoblast activity. The method then further comprises additionallyadministering, either (1) contemporaneous with, (b) overlapping with, or(3) subsequent to the administration of the bone anabolic agent, anantiresorptive agent for a duration and at a concentration sufficient tosubstantially prevent resorption of new bone produced due to theincreased osteoblast activity achieved in accordance with the invention.As with the method described above, the mechanical alteration of thecontents of the bone marrow cavity thus permits specific bones of thesubject to be targeted for enhanced bone formation.

In one embodiment of the above-described method the bone anabolic agentmay be selected from the group consisting of natural parathyroidhormone, a truncate of natural parathyroid hormone, an amidated truncateof natural parathyroid hormone, an amidated natural parathyroid hormoneand combinations thereof. In one particular embodiment, the agent may bePTH-[1-34]in the free acid form, sold by Eli Lilly & Co. under the trademark FORTEO® (teriparatide). In a further embodiment the bone anabolicagent is an amidated truncate of natural parathyroid hormone and may beselected from among PTH[1-30]NH₂, PTH[1-31]NH₂, PTH[1-32]NH₂,PTH[1-33]NH₂, PTH[1-34]NH₂ and combinations thereof. A preferred choicefor the bone anabolic agent is PTH[1-34]N₂.

In a further embodiment of the invention a sufficient amount of anamidated truncate of natural parathyroid hormone is administered to thesubject to achieve a pulsatile blood concentration thereof in thesubject of between about 50 and 500 pg/ml, preferably between about100-200 pg/ml and most preferably about 150 pg/ml. In the case of aninjectable formulation, for example, the dose (in pure weight of theactive hormone) given to a human subject, may be that taught in theliterature relating to the bone anabolic activity of these variousagents. Such dose may, but does not necessarily, range between about10-200 μg per dose, more preferably between about 20-100 μg and mostpreferably between about 20-50 μg. When alternate delivery methods areused the dose may, but does not necessarily, range between about 10μg-10 mg.

In a particular embodiment the antiresorptive agent is a calcitoninselected from among human calcitonin, salmon calcitonin, eel calcitonin,elkatonin, porcine calcitonin, chicken calcitonin gene related peptide(CGRP) and combinations thereof. In one embodiment the antiresorptiveagent is salmon calcitonin. It is preferred to achieve blood levels ofsalmon calcitonin, when it is used as the antiresorptive agent, ofbetween about 5-500 pg/ml, more preferably between about 10-250 pg/mland most preferably between about 20-50 pg/ml. To achieve theabove-indicated serum levels daily dosages of the antiresorptive agentused in, e.g., injectable formulations, may range between about 5-200 μg(pure weight of the drug), more preferably between about 5-50 μg andmost preferably between about 8-20 μg. When alternate delivery methods,i.e., other than injection, are used, the dose may range between about 5μg-5 mg. Administration of the antiresorptive agent preferably continuesfor at least 3 months and more preferably between 12-24 months.

One embodiment of the invention comprises the use of any of the abovedescribed methods to form a sufficient amount of additional bone in ajaw region of the subject to provide an anchor for a dental implantimplanted therein. Alternately, or additionally, any of the methodsdescribed above may be utilized to form a sufficient amount ofadditional bone in one or more targeted bones of the subject to permitsecure anchoring of a prosthetic device thereto. Such prosthetic devicesmay include, but are not limited to, a prosthetic knee, shoulder or hip.In a further embodiment, any of the methods of the invention may beutilized to form a sufficient amount of additional bone at any locationin the subject to serve as a secure anchor for a hollow, adjustableinsert anchored thereto. Additionally, any of the methods of theinvention may be used in targeting at least one vertebra for additionalbone formation, wherein a sufficient amount of bone is added to the atleast one vertebra such that the subject is substantially freed fromchronic pain caused due to vertebral crush.

In a still further embodiment the invention provides a kit for fosteringbone formation in at least one targeted bone of a subject in need ofsuch bone formation. The kit comprises at least one container havingtherein at least one bone anabolic agent and a mechanical alterationdevice for altering contents of a bone marrow cavity in at least onetargeted bone of a subject. In another embodiment the kit mayadditionally comprise an evacuation device for evacuating at least aportion of the contents from the bone marrow cavity. In a furtherembodiment the kit may additionally comprise at least one containerhaving therein at least one antiresorptive agent.

In one embodiment of the kit of the invention, the bone anabolic agentis selected from among natural parathyroid hormone, a truncate ofnatural parathyroid hormone, an amidated truncate of natural parathyroidhormone, an amidated natural parathyroid hormone, and combinationsthereof. In a preferred embodiment the bone anabolic agent is a truncateof natural parathyroid hormone. A preferred truncate for use as theagent is PTH[1-34] in the free acid form. Other preferred truncatesinclude amidated truncates. The bone anabolic agent may thus be selectedfrom among PTH[1-30]NH₂, PTH[1-31]NH₂, PTH[1-32]NH₂, PTH[1-33]NH₂,PTH[1-34]NH₂ and combinations thereof. In a specific embodiment the boneanabolic agent is PTH[1-34]NH₂.

In an additional embodiment of the kit of the invention theantiresorptive agent is a calcitonin selected from the group consistingof human calcitonin, salmon calcitonin, eel calcitonin, elkatonin,porcine calcitonin, chicken calcitonin, calcitonin related gene peptide(CGRP) and combinations thereof. In a particular embodiment theantiresorptive agent is salmon calcitonin.

Although the present invention has been described in relation toparticular embodiments thereof, many other variations and modificationsand other uses will become apparent to those skilled in the art. Thepresent invention, therefore, is not limited by the specific disclosureherein, but only by the claims.

1. A method of medical treatment producing a targeted increase in bonestrength, form or volume in a subject in need thereof, which methodcomprises the steps of: (a) mechanically inducing an increase inosteoblast activity in said subject by a surgical treatment of at leastone bone of said subject; and (b) elevating blood concentration of atleast one bone anabolic agent in said subject, wherein steps (a) and (b)are performed in any order, but in sufficient time proximity that saidelevated concentration of said anabolic agent and said mechanicallyinduced increase in osteoblast activity at least partially overlaps. 2.The method of claim 1, wherein the blood concentration of the boneanabolic agent is elevated by direct administration of a bone anabolicagent to said subject.
 3. The method of claim 1, which further comprisestargeting one or more specific bones of said subject for said inducedbone growth by mechanically altering the contents of a bone marrowcavity within each said targeted bone so as to induce said increasedosteoblast activity therein.
 4. The method of claim 1, which furthercomprises providing said subject with an elevated blood concentration ofat least one antiresorptive agent, wherein said elevated concentrationis sufficient to substantially reduce resorption of new bone growthproduced due to said increased osteoblast activity.
 5. The method ofclaim 1 additionally comprising forming a sufficient amount ofadditional bone in a jaw region of said subject to provide an anchor fora dental implant implanted into said jaw region.
 6. The method of claim1 additionally comprising forming a sufficient amount of additional bonein one or more targeted bones of said subject to permit a prostheticdevice implanted into at least one said targeted bone to be securelyanchored thereto.
 7. The method of claim 1, additionally comprisingforming a sufficient amount of additional bone in said subject to serveas a secure anchor for a hollow, adjustable insert anchored to saidadditional bone.
 8. The method of claim 1, which further comprisestargeting at least one vertebra of said subject for additional boneformation and wherein a sufficient amount of bone is added to said atleast one vertebra such that the subject is substantially freed fromchronic pain caused due to vertebral crush.
 9. The method of claim 1,wherein additional bone is formed on at least one vertebra of saidsubject in an amount sufficient to stabilize said at least one vertebradue to strengthening thereof.
 10. The method of claim 1 wherein themechanical induction of osteoblast activity is accomplished byinserting, into the bone marrow cavity of a bone targeted for enhancedbone formation, an object configured or adapted to physically alter thecontents of said cavity and thereby to stimulate said osteoblastactivity within said cavity.
 11. The method of claim 10, wherein saidphysical alteration further comprises removal of at least a portion ofsaid altered cavity contents to provide additional room in the cavityfor increased bone mass.
 12. The method of claim 1, wherein said atleast one bone anabolic agent is selected from the group consisting ofparathyroid hormone (PTH), anabolic Vitamin D analogs, a low-densitylipoprotein receptor-related protein 5 (LRP5), an activator ofnon-genomic estrogen-like signaling (ANGELS), a bone morphogenic protein(BMP), an insulin-like growth factor (IGF), a fibroblast growth factor(FGF), sclerostin, leptin, a prostaglandin, a statin, strontium, agrowth hormone, a growth hormone releasing factor (GHRF), hepatocytegrowth factor (HGF), calcitonin gene related peptide (CGRP), parathyroidhormone related peptide (PTHrP), transforming growth factor (TGF)-β1 andcombinations thereof.
 13. The method of claim 12, wherein the boneanabolic agent is at least one parathyroid hormone selected from thegroup consisting of natural parathyroid hormone, a truncate of naturalparathyroid hormone, an amidated truncate of natural parathyroidhormone, an amidated natural parathyroid hormone, and combinationsthereof.
 14. The method of claim 13, wherein the bone anabolic agent isselected from the group consisting of PTH[1-84] in the free acid form,PTH[1-84]NH₂, PTH [1-34] in the free acid form, PTH[1-30]NH₂,PTH[1-31]NH₂, PTH[1-32]NH₂, PTH[1-33]NH₂, PTH[1-34]NH₂ and combinationsthereof.
 15. The method of claim 13, wherein a sufficient amount of saidparathyroid hormone is administered to said subject to achieve apulsatile blood concentration thereof in said subject of between about50-350 pg/ml.
 16. The method of claim 15, wherein said sufficient amountof parathyroid hormone is from about 10 μg-10 mg pure weight of PTHhormone per dose.
 17. The method of claim 15, wherein said parathyroidhormone is administered via injection and the sufficient amount ofparathyroid hormone is from about 10-200 μg per dose.
 18. The method ofclaim 15, wherein the blood concentration of said parathyroid hormone insaid subject is raised to a level of between about 50-350 pg/ml by nolater than 7 days following mechanical alteration of the contents of thebone marrow cavity.
 19. The method of claim 1 which further comprises astep of: (c) administering to said subject an antiresorptive agent for atime and at a concentration sufficient to substantially reduceresorption of the new bone produced due to said osteoblast activity,wherein steps (b) and (c) are performed in any order, but in sufficienttime proximity that said elevated concentration of said anabolic agentand said mechanically induced increase in osteoblast activity by asurgical treatment of at least one bone of said subject, at leastpartially overlaps.
 20. The method of claim 19, wherein administrationof said antiresorptive agent is commenced during administration of thebone anabolic agent and continues beyond termination of said boneanabolic agent administration.
 21. The method of claim 19, wherein theantiresorptive agent is administered orally, intravenously,intramuscularly, subcutaneously, via implant, transmucosally, rectally,nasally, by depot injection, by inhalation and pulmonary absorption ortransdermally.
 22. The method of claim 19, wherein the antiresorptiveagent is a calcitonin selected from the group consisting of humancalcitonin, salmon calcitonin, eel calcitonin, elkatonin, porcinecalcitonin, chicken calcitonin, calcitonin gene related peptide (CGRP)and combinations thereof.
 23. The method of claim 22, wherein theantiresorptive agent is salmon calcitonin and wherein the salmoncalcitonin is administered to said subject in an amount calculated toachieve a substantially continuous blood concentration thereof ofbetween about 5-500 pg/ml.
 24. The method of claim 23, wherein theamount of said salmon calcitonin is from about 5 μg to 5 mg pure weightof the calcitonin per dose.
 25. The method of claim 23, wherein thesalmon calcitonin is administered via injection and the amount of saidsalmon calcitonin is from about 5 μg -200 μg per dose.
 26. A medicaltreatment for inducing bone formation in a subject in need of suchinducement, comprising the steps of: (a) mechanically inducing anincrease in osteoblast activity in said subject by a surgical treatmentof at least one bone of said subject; and (b) administering to saidsubject at least one agent that causes elevated blood levels of anendogenous bone anabolic agent within said subject, wherein steps (a)and (b) are performed in any order, but in sufficient time proximitythat said elevated concentration of said anabolic agent and saidmechanically induced increase in osteoblast activity at least partiallyoverlaps.
 27. The method of claim 26, wherein the agent causing anincreased expression of said endogenous bone anabolic agent within saidsubject is a calcilytic agent.